Antibodies to train R: a cysteine-rich member of the TNF-receptor family, and methods of treating tumors expressing said receptor

ABSTRACT

Embodiments of the present invention encompass novel receptors of the TNF family. In particular, embodiments of the invention encompass TNF Receptor in the BRAIN (TRAIN) and isolated antibodies that bind to the same. In some embodiments, isolated antibodies that bind to TRAIN are useful in regulating cell differentiation, death, and survival. Other embodiments of the invention encompass pharmaceutical compositions comprising antibodies that bind to human TRAIN.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 11/396,907,filed Apr. 4, 2006, now issued U.S. Pat. No. 7,402,658, which is acontinuation of application Ser. No. 10/303,502, filed Nov. 22, 2002,now issued U.S. Pat. No. 7,223,852, which is a continuation ofapplication Ser. No. 09/522,436, filed Mar. 9, 2000, now abandoned,which is a continuation-in-part of International Application No.PCT/US98/19030, filed Sep. 11, 1998, which claims the benefit ofapplication Ser. No. 60/084,422, filed May 6, 1998 and the benefit ofapplication Ser. No. 60/058,631, filed Sep. 12, 1997, all of which areincorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

The present invention relates to novel receptors in the TNF family. Anovel receptor has been identified, referred to herein as TRAIN (TNFReceptor in the BRAIN).

The TNF family consists of pairs of ligands and their specific receptorsreferred to as TNF family ligands and TNF family receptors (Bazzoni andBeutler, 1996. N Engl J Med 334, 1717-25). The family is involved in theregulation of the immune system and possibly other non-immunologicalsystems. The regulation is often at a “master switch” level such thatTNF family signaling can result in a large number of subsequent eventsbest typified by TNF. TNF can initiate the general protectiveinflammatory response of an organism to foreign invasion that involvesthe altered display of adhesion molecules involved in cell trafficking,chemokine production to drive specific cells into specific compartmentsand the priming of various effector cells. As such, the regulation ofthese pathways has clinical potential.

The TNF receptor family is a collection of related proteins thatgenerally consist of an extracellular domain, a transmembrane domain andan intracellular signaling domain. The extracellular domain is builtfrom 2-6 copies of a tightly disulphide bonded domain and is recognizedon the basis of the unique arrangement of cysteine residues. Eachreceptor binds to a corresponding ligand although one ligand may shareseveral receptors. In some cases, it is clear that by alternate RNAsplicing, soluble forms of the receptors lacking the transmembraneregion and intracellular domain exist naturally. Moreover, in nature,truncated versions of these receptors exist and the soluble inhibitoryform may have direct biological regulatory roles. Clearly, viruses haveused this tactic to inhibit TNF activity in their host organisms (Smith,1994. Trends in Microbiol. 82, 81-88). These receptors can signal anumber of events including cell differentiation, cell death or cellsurvival signals. Cell death signaling often is triggered via relativelydirect links to the caspase cascade of proteases e.g. Fas and TNFreceptors. Most receptors in this class can also activate NFKBcontrolled events.

An emerging theme in the TNF family of receptors has been the use bynature of both full length receptors with intracellular domains thattransmit a signal and alternate forms which are either secreted or lackan intracellular signaling domain. These later forms can inhibit ligandsignaling and hence can dampen a biological response. There are severalexamples of this phenomenon. First, the TNF receptor p75 is readilysecreted following selective cleavage from the membrane and then acts toblock the action of TNF. It is likely that nature has evolved thissystem to buffer TNF activity. A second example is provided by theTRIAL-TRAIL receptor system where there are 4 separate genes encodingTRAIL receptors. Two of these TRAIL-R1 and TRAIL-R2 possessintracellular domains and transduce signal. A third receptor (TRAIL-R4)has an intracellular domain yet this domain does not have all theelements is found in R1 and R2, e.g. it lacks a domain capable ofsignaling cell death. Lastly, there is a fourth receptor TRAIL-R3, thatis essentially a soluble form but remains tethered by a glycolipidlinkage. Hence this receptor can bind ligand yet it is unable totransmit a signal, i.e. it is effectively a decoy receptor. A thirdexample is provided by the osteoprotegerin (OPG) system where the OPGreceptor lacks a transmembrane domain and is secreted into the medium.This receptor can block the signaling necessary to induce osteoclastdifferentiation possibly by binding to a ligand called RANK-L. The TRAINsystem described here resembles the OPG paradigm in that a short versioncan be secreted that would inhibit the natural TRAIN-L (currentlyunknown) from binding to full length TRAIN and eliciting a signal.

The receptors are powerful tools to elucidate biological pathways viatheir easy conversion to immunoglobulin fusion proteins. These dimericsoluble receptor forms are good inhibitors of events mediated by eithersecreted or surface bound ligands. By binding to these ligands theyprevent the ligand from interacting with cell associated receptors thatcan signal. Not only are these receptor-Ig fusion proteins useful in anexperimental sense, but they have been successfully used clinically inthe case of TNF-R-Ig to treat inflammatory bowel disease, rheumatoidarthritis and the acute clinical syndrome accompanying OKT3administration (Eason et al., 1996. Transplantation 61, 224-8; Feldmannet al., 1996. Annu Rev Immunol; van Dullemen et al., 1995.Gastroenterology 109, 129-35). One can envision that manipulation of themany events mediated by signaling through the TNF family of receptorswill have wide application in the treatment of immune based diseases andalso the wide range of s human diseases that have pathological sequelaedue to immune system involvement. A soluble form of a recently describedreceptor, osteoprotegerin, can block the loss of bone mass and,therefore, the events controlled by TNF family receptor signaling arenot necessarily limited to immune system regulation. Antibodies to thereceptor can block ligand binding and hence can also have clinicalapplication. Such antibodies are often very long-lived and may haveadvantages over soluble receptor-Ig fusion proteins which have shorterblood half-lives.

While inhibition of the receptor mediated pathway represents the mostexploited therapeutic application of these receptors, originally it wasthe activation of the TNF receptors that showed clinical promise(Aggarwal and Natarajan, 1996. Eur Cytokine Netw 7, 93-124). Activationof the TNF receptors can initiate cell death in the target cell andhence the application to tumors was and still is attractive (Eggermontet al., 1996. J Clin Oncol 14, 2653-65). The receptor can be activatedeither by administration of the ligand, i.e. the natural pathway or someantibodies that can crosslink the receptor are also potent agonists.Antibodies would have an advantage in oncology since they can persist inthe blood for long periods whereas the ligands generally have shortlifespans in the blood. As many of these receptors may be expressed moreselectively in tumors or they may only signal cell death ordifferentiation in tumors, agonist antibodies could be good weapons inthe treatment of cancer. Likewise, many positive immunological eventsare mediated via the TNF family receptors, e.g. host inflammatoryreactions, antibody production etc. and therefore agonistic antibodiescould have beneficial effects in other, non-oncological applications.

Paradoxically, the inhibition of a pathway may have clinical benefit inthe treatment of tumors. For example the Fas ligand is expressed by sometumors and this expression can lead to the death of Fas positivelymphocytes thus facilitating the ability of the tumor to evade theimmune system. In this case, inhibition of the Fas system could thenallow the immune system to react to the tumor in other ways now thataccess is possible (Green and Ware, 1997. Natl. Acad. Sci. USA 94,5986-5990).

The receptors are also useful to discover the corresponding ligand asthey can serve as probes of the ligand in expression cloning techniques(Smith et al., 1993. Cell 73, 1349-60). Likewise, the receptors andligands can form in vitro binding assays that will allow theidentification of inhibitory substances. Such substances can form thebasis of novel inhibitors of the pathways.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide sequence for human TRAIN receptor (SEQ IDNO: 7) from a composite of two lambda gt10 clones (GJ159 and GJ158).

FIG. 2 shows a comparison of human TRAIN receptor (top) (amino acids1-214 of SEO ID NO: 3) and murine TRAIN receptor long (bottom) (SEQ IDNO: 2).

FIG. 3 shows the nucleotide sequences for human TRAIN receptor from asubclone of lambda gt10 cDNA (SEQ ID NO: 8).

FIG. 4 shows the amino acid sequence for human TRAIN (SEQ ID NO: 3)corresponding to the nucleotide sequence in FIG. 1.

FIG. 5 shows the amino acid sequence for human TRAIN (SEQ ID NO: 4)corresponding to the nucleotide sequence in FIG. 3.

A. DEFINITIONS

“Homologous”, as used herein, refers to the sequence similarity betweensequences of molecules being compared. When a position in both of thetwo compared sequences is occupied by the same base or amino acidmonomer subunit, e.g., if a position in each of two DNA molecules isoccupied by adenine, then the molecules are homologous at that position.The percent of homology between two sequences is a function of thenumber of matching or homologous positions shared by the two sequencesdivided by the number of positions compared×100. For example, if 6 of 10of the positions in two sequences are matched or homologous then the twosequences are 60% homologous. By way of example, the DNA sequencesATTGCC and TATGGC share 50% homology. Generally, a comparison is madewhen two sequences are aligned to give maximum homology.

A “purified preparation” or a “substantially pure preparation” of apolypeptide, as used herein, means a polypeptide that has been separatedfrom other proteins, lipids, and nucleic acids with which it naturallyoccurs. Preferably, the polypeptide is also separated from othersubstances, e.g., antibodies, matrices, etc., which are used to purifyit.

“Transformed host” as used herein is meant to encompass any host withstably integrated sequence, i.e. TRAIN sequence, introduced into itsgenome or a host possessing sequence, i.e. receptor encoding episomalelements.

A “treatment”, as used herein, includes any therapeutic treatment, e.g.,the administration of a therapeutic agent or substance, e.g., a drug.

A “substantially pure nucleic acid”, e.g., a substantially pure DNA, isa nucleic acid which is one or both of: (1) not immediately contiguouswith either one or both of the sequences, e.g., coding sequences, withwhich it is immediately contiguous (i.e., one at the 5′ end and one atthe 3′ end) in the naturally-occurring genome of the organism from whichthe nucleic acid is derived; or (2) which is substantially free of anucleic acid sequence with which it occurs in the organism from whichthe nucleic acid is derived. The term includes, for example, arecombinant DNA which is incorporated into a vector, e.g., into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g., acDNA or a genomic DNA fragment produced by PCR or restrictionendonuclease treatment) independent of other DNA sequences.Substantially pure DNA also includes a recombinant DNA which is part ofa hybrid gene encoding TRAIN.

The terms “peptides”, “proteins”, and “polypeptides” are usedinterchangeably herein.

“Biologically active” as used herein, means having an in vivo or invitro activity which may be performed directly or indirectly.Biologically active fragments of TRAIN may have, for example, 70% aminoacid homology with the active site of the receptor, more preferably atleast 80%, and most preferably, at least 90% homology. Identity orhomology with respect to the receptor is defined herein as thepercentage of amino acid residues in the candidate sequence which areidentical to the TRAIN residues in SEQ. ID. NO. 3.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare described in the literature.

The claimed invention relates to a novel receptor designated TRAIN-R.The amino acid sequence of murine TRAIN-R is set forth in SEQ. I). NO. 1(the short form) and SEQ. ID. NO. 2, (the long form). The full lengthamino acid sequence of human TRAIN-R is set forth in SEQ. ID. NO. 3 andFIG. 1. As shown in FIG. 1, the protein length is 417 amino acids. Thepredicted signal sequence runs from residues 1-25. It is believed thatthe mature N-terminus is at amino acid residue 26, the extracellulardomain spans residues 26-173, the transmembrane domain spans residues174-190, and the cytoplasmic domain spans residues 191-417. There is apotential N-linked glycosylation site at residue 105.

SEQ. ID. NO. 4 sets forth the amino acid sequence for the carboxyterminal 30 amino acids of a secreted form of human TRAIN-R from asubclone of lambda gt10 cDNA (GJ156). This peptide sequence features 30amino acids that are identical to amino acids 121-149 of the compositeprotein shown in FIG. 1 and are identical to amino acids 121-150 of theC-terminus of murine TRAIN-R short form (secreted protein). SEQ. ID. NO9 shows the amino acid sequence of the entire short secreted form of thehuman TRAIN-R based on the alternate cloned exon and by comparison tothe mouse short form.

FIG. 2 shows a comparison of the first 214 amino acids of human TRAIN-R(417 a.a.) and murine TRAIN-R long (214 a.a.). As shown in the Figure,the two sequences have an identity of about 81.8%.

The TRAIN receptors of the invention may be isolated from mammaliantissues and purified to homogeneity, or isolated from cells whichcontain membrane-bound TRAIN-R, and purified to homogeneity. Methods forgrowing cells and isolating cell extracts are well known in the art, asare various cell types and growth and isolation methods. In general, anyTRAIN-R can be isolated from any cell or tissue expressing this proteinusing a cDNA probe, isolating MRNA and transcribing the mRNA into cDNA.Thereafter, the protein can be produced by inserting the cDNA into anexpression vector, such as a virus, plasmid, cosmid or other expressionvector, inserting the expression vector into a cell, and proliferatingthe resulting cells. The TRAIN-R can then be isolated from the medium orcell extract by methods well known in the art. One skilled in the artcan readily vary the vectors and cell lines and still obtain the claimedreceptors. Alternatively, TRAIN receptors can be chemically synthesizedusing the sequences set forth in SEQ. ID. NOs. 1, 2, 3 or 4.

It is believed that murine TRAIN-R is expressed highest in brain andlung and at a lower level in liver, skeletal muscle and kidney. Theexpression pattern of human TRAIN-R differs in that a low level ofexpression has been detected in every tissue and cell line tested thusfar (ubiquitous) with a significantly higher expression detected inheart, prostate, ovary, testis, peripheral blood lymphocytes (PBLs),thyroid, and adrenal gland.

Murine TRAIN-R may exist in nature as a natural soluble form asindicated in SEQ. ID. NO. 1. Human TRAIN-R may exist as a naturalsoluble form having the carboxy sequence indicated in SEQ. ID. NO. 4 andFIG. 3. The soluble protein should inhibit signaling by the full lengthTRAIN-R.

The present invention also encompasses DNA sequences which encode themurine (both long and short) and human TRAIN receptors (full length andcarboxy terminus). These DNA sequences are set forth in SEQ. ID. NOs. 5,6, 7 and 8, respectively. The human TRAIN-R sequence in SEQ. ID. NO. 7contains 5′UTR, a complete coding region, a stop codon and some 3′UTR.FIG. 1 shows the nucleotide sequence for human TRAIN-R as derived from acomposite sequence of GJ159 and GJ158. As shown in FIG. 1, human TRAIN-Rhas a nucleotide sequence length of 2185, a coding region from 179-1429,and a stop codon at 1430-1432.

The human TRAIN-R sequence in SEQ. ID. NO. 8 contains intron sequence,an exon encoding the carboxy terminal 30 amino acids of a secreted formof human TRAIN-R, a stop codon and 3′UTR. As shown in FIG. 3, it isbelieved that the intron is at residues 1-350, the coding region at352-441, the stop codon at 442-444 and the 3′UTR=445-791.

In other embodiments, the invention relates to sequences that have atleast 50% homology with DNA sequences encoding the C terminal receptorbinding domain of the ligands and hybridize to the claimed DNA sequencesor fragments thereof, and which encode the TRAIN receptors having thesequences identified in SEQ. ID. NO. 1, 2,3 or4.

The invention in certain embodiments furthermore relates to DNAsequences encoding the TRAIN receptors where the sequences areoperatively linked to an expression control sequence. Any suitableexpression control sequences are useful in the claimed invention, andcan easily be selected by one skilled in the art.

The invention also contemplates recombinant DNAs comprising a sequenceencoding TRAIN receptors or fragments thereof, as well as hosts withstably integrated TRAIN-R sequences introduced into their genome, orpossessing episomal elements. Any suitable host may be used in theinvention, and can easily be selected by one skilled in the art withoutundue experimentation.

The claimed invention in certain embodiments encompasses recombinantTRAIN-R. One skilled in the art can readily isolate such recombinantreceptors thereby providing substantially pure recombinant TRAIN-Rpolypeptides. Isolated receptors of the invention are substantially freeof other contaminating materials of natural or endogenous origin, andcontain less than about 10-15% by mass of protein contaminants residualof production processes.

Mammalian Receptors within the scope of the invention also include, butare not limited to, primate, human, murine, canine, feline, bovine,ovine, equine and porcine TRAIN-R. Mammalian Receptors can also beobtained by cross species hybridization using a single stranded cDNAderived from the human TRAIN-R. DNA sequences of the invention can beused as a hybridization probe to isolate Receptor cDNAS from othermammalian cDNA libraries.

Derivatives of the Receptors within the scope of the invention alsoinclude various structural forms of the proteins of SEQ. ID. NOs. 1, 2,3 and 4 which retain biological activity. For example, a receptorprotein may be in the form of acidic or basic salts, or may be inneutral form. Individual amino acid residues may also be modified byoxidation or reduction.

Receptor derivatives may also be used as immunogens, reagents in areceptor-based immunoassay, or as binding agents for affinitypurification procedures of TRAIN ligands.

The present invention also includes TRAIN-R with or without associatednative-pattern glycosylation. One skilled in the art will understandthat the glycosylation pattern on the receptor may vary depending on theparticular expression system used. For example, typically, expression inbacteria such as E. coli results in a non-glycosylated molecule. TRAIN-Rderivatives may also be obtained by mutations of the receptors or theirsubunits. A mutant, as referred to herein, is a polypeptide homologousto a claimed Receptor but which has an amino acid sequence differentfrom the native sequence due to a deletion, insertion or substitution.

Bioequivalent analogs of the Receptor proteins of the invention may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, often cysteine residues canbe deleted or replaced with other amino acids to prevent formation ofunnecessary or incorrect intramolecular disulfide bridges uponrenaturation. Other approaches to mutagenesis involved modifications,for example, to enhance expression in the chosen expression system.

Soluble Receptors of the invention may comprise subunits which have beenchanged from a membrane bound to a soluble form. Thus, soluble peptidesmay be produced by truncating the polypeptide to remove, for example,the cytoplasmic tail and/or transmembrane region. Alternatively, thetransmembrane domain may be inactivated by deletion, or by substitutionsof the normally hydrophobic amino acid residues which comprise atransmembrane domain with hydrophilic ones. In either case, asubstantially hydrophilic hydropathy profile is created which willreduce lipid affinity and improve aqueous solubility. Deletion of thetransmembrane domain is preferred over substitution with hydrophilicamino acid residues because it avoids introducing potentiallyimmunogenic epitopes. Soluble Receptors of the invention may include anynumber of well-known leader sequences at the N-terminus. Such a sequencewould allow the peptides to be expressed and targeted to the secretionpathway in a eukaryotic system.

The invention herein provides agents, such as agonists and antagonists,directed against the claimed receptors. In certain embodiments of thisinvention, the agent comprises a blocking agent that comprises andantibody directed against the TRAIN-R that inhibits TRAIN receptorsignaling. Preferably the antibody is a monoclonal antibody. Similarly,the claimed invention encompasses antibodies and other agents which actas agonists in the TRAIN pathways.

Inhibitory anti-TRAIN-R antibodies and other receptor blocking agentscan be identified using screening methods that detect the ability of oneor more agents either to bind to the TRAIN-R, or ligands thereto, or toinhibit the effects of TRAIN-R signaling on cells.

One skilled in the art will have knowledge of a number of assays thatmeasure the strength of ligand-receptor binding and can be used toperform competition assays with putative TRAIN receptor blocking agents.The strength of the binding between a receptor and ligand can bemeasured using an enzyme-linked immunoadsorption assay (ELISA) or aradioimmunoassay (RIA). Specific binding may also be measured byflourescently labeling antibody-antigen complexes and performingfluorescence activated cell sorting analysis (FACS), or by performingother such immunodetection methods, all of which are techniqueswell-known in the art.

With any of these or other techniques for measuring receptor-ligandinteractions, one skilled in the art can evaluate the ability of ablocking agent, alone or in combination with other agents, to inhibitbinding of ligands to the receptor molecules. Such assays may also beused to test blocking agents or derivatives of such agents, i.e.fusions, chimeras, mutants or chemically altered forms, to optimize theability of the agent to block receptor activation.

The receptor blocking agents of the invention in one embodiment comprisesoluble TRAIN receptor molecules. Using the sequence information hereinand recombinant DNA techniques well known in the art, functionalfragments encoding the TRAIN receptor ligand binding domain can becloned into a vector and expressed in an appropriate host to produce asoluble receptor molecule. Soluble TRAIN receptor molecules that cancompete with native TRAIN receptors for ligand binding according to theassays described herein can be selected as TRAIN receptor blockingagents.

A soluble TRAIN receptor comprising amino acid sequences selected formthose shown herein may be attached to one or more heterologous proteindomains (“fusion domains”) to increase the in vivo stability of thereceptor fusion protein, or to modulate its biological activity orlocalization.

Preferably, stable plasma proteins—which typically have a half-lifegreater than 20 hours in the circulation of a mammal—are used toconstruct the receptor fusion proteins. Such plasma proteins include butare not limited to: immunoglobulins, serum albumin, lipoproteins,apolipoproteins and transferrin. Sequences that can target the solublereceptors to a particular cell or tissue type may also be attached tothe receptor ligand binding domain to create a specifically localizedsoluble receptor fusion protein.

All or a functional fragment of the TRAIN receptor extracellular regioncomprising the TRAIN receptor ligand binding domain may be fused to animmunoglobulin constant region like the Fc domain of a human IgG1 heavychain. Soluble receptor-IgG fusions proteins are common immunologicalreagents and methods for their construction are well known in the art.(see, e.g. U.S. Pat. No. 5,225,538).

A functional TRAIN-R ligand binding domain may be fused to animmunoglobulin (Ig) Fc domain derived from an immunoglobulin class orsubclass other than IgG1. The Fc domains of antibodies belonging todifferent Ig classes or subclasses can activate diverse secondaryeffector functions. Activation occurs when the Fc domain is bound by acognate Fc receptor. Secondary effector functions include the ability toactivate the complement system, to cross the placenta and to bindvarious microbial proteins. The properties of the different classes andsubclasses of immunoglobulins are described in the art.

Activation of the complement system initiates cascades of enzymaticreactions that mediate inflammation. The products of the complementsystem have a variety of functions, including binding of bacteria,endocytosis, phagocytosis, cytotoxicity, free radical production andsolubilization of immune complexes.

The complement enzyme cascade can be activated by the Fc domains ofantigen-bound IgG1, IgG3 and Ig M antibodies. The Fc domain of IgG2appears to be less effective, and the Fc domains of IgG4, IgA, IgD andIgE are ineffective at activating complement. Thus one can select an Fcdomain based on whether its associated secondary effector functions aredesirable for the particular immune response or disease being treatedwith the receptor-fusion protein.

It if would be advantageous to harm or kill the TRAIN ligand bearingtarget cell, one could, for example, select an especially active Fcdomain (IgG1) to make the fusion protein. Alternatively, if it would bedesirable to target the TRAIN receptor-FC fusion to a cell withouttriggering the complement system, an inactive IgG4 Fc domain could beselected.

Mutations in Fc domains that reduce or eliminate binding to Fc receptorsand complement activation have been described in the art. These or othermutations can be used, alone or in combination to optimize the activityof the Fc domain used to construct the TRAIN receptor-Fc fusion protein.

One skilled in the art will appreciate that different amino acidresidues forming the junction point of the receptor-Ig fusion proteinmay alter the structure, stability and ultimate biological activity ofthe soluble TRAIN receptor fusion protein. One or more amino acids maybe added to the C-terminus of the selected TRAIN receptor fragment tomodify the junction point whit the selected fusion domain.

The N-terminus of the TRAIN receptor fusion protein may also be variedby changing the position at which the selected TRAIN receptor DNAfragment is cleaved at its 5′ end for insertion into the recombinantexpression vector. The stability and activity of each TRAIN receptorfusion protein may be tested and optimized using routine experimentationand the assays for selecting blocking agents described herein.

Using the TRAIN receptor binding domain sequences within theextracellular domain as shown herein, amino acid sequence variants mayalso be constructed to modify the affinity of the soluble TRAIN receptormolecules for their ligands. The soluble molecules of this invention cancompete for binding with endogenous receptors. It is envisioned that anysoluble molecule comprising a TRAIN receptor ligand binding domain thatcan compete with native receptors for ligand binding is a receptorblocking agent that falls within the scope of the present invention.

In other embodiments of this invention, antibodies directed against theTRAIL and TRAIN receptors (anti-TRAIN-R abs) function as receptorblocking agents. The antibodies of this invention can be polyclonal ormonoclonal and can be modified to optimize their ability to blockTRAIN-R signaling, their bioavailability, stability or other desiredtraits.

Polyclonal antibody sera directed against TRAIN-R are prepared usingconventional techniques by injecting animals such as goats, rabbits,rats, hamsters or mice subcutaneously with TRAIN-R-Fc fusion protein inFreund's adjuvant, followed by booster intraperitoneal or subcutaneousinjection in incomplete Freund's. Polyclonal antisera containing thedesired antibodies directed against the TRAIN receptors can then bescreened by conventional immunological procedures.

Various forms of anti-TRAIN-R abs can also be made using standardrecombinant DNA techniques. For example, “chimeric” antibodies can beconstructed in which the antigen binding domain from an animal antibodyis linked to a human constant domain. Chimeric antibodies reduce theobserved immunogenic responses elicited by animal antibodies when usedin human clinical treatments.

In addition, recombinant “humanized” antibodies which can recognize theTRAIN-R can be synthesized. Human antibodies are chimeras comprisingmostly human IgG sequences into which the regions responsible forspecific antigen-binding have been inserted. (e.g. WO 94/04679). Animalsare immunized with the desired antigen, the corresponding antibodies areisolated, and the portion of the variable region sequences responsiblefor specific antigen binding are removed. The animal-derived antigenbinding regions are then cloned into the appropriate position of humanantibody genes in which the antigen binding regions have been deleted.Humanized antibodies minimize the use of heterologous (inter species)sequences in human antibodies, and are less likely to elicit immuneresponses in the mammal being treated.

Construction of different classes of recombinant anti-TRAIN-R antibodiescan also be accomplished by making chimeric or humanized antibodiescomprising the anti-R variable domains and human constant domainsisolated from different classes of immunoglobulins. For example,anti-TRAIN-R IgM antibodies with increased antigen binding sitevalencies can be recombinantly produced by cloning the antigen bindingsite into vectors carrying the human μ chain constant regions.

In addition, standard recombinant DNA techniques can be used to alterthe binding affinities of recombinant antibodies with their antigens byaltering amino acid residues in the vicinity of the antigen bindingsites. The antigen binding affinity of a humanized antibody can beincreased by mutagenesis based on molecular modeling.

It may be desirable to increase or decrease the affinity of anti-TRAIN-Rantibodies for the receptors depending on the targeted tissue type orthe particular treatment schedule envisioned. For example, it may beadvantageous to treat a patient with constant levels of anti-Receptorantibodies with reduced ability to signal through the pathway forsemi-prophylactic treatments. Likewise, inhibitory anti-TRAIN-Rantibodies with increased affinity for the receptors may be advantageousfor short term treatments.

The claimed invention in yet other embodiments encompassespharmaceutical compositions comprising an effective amount of a TRAIN-Rblocking or activating agent, and pharmaceutically acceptable carriers.The compositions of the invention will be administered at an effectivedose to treat the particular clinical condition addressed. Determinationof a preferred pharmaceutical formulation and a therapeuticallyefficient dose regiment for a given application is well within the skillof the art taking into consideration for example, the condition andweight of the patient, the extent of desired treatment and the toleranceof the patient for the treatment. Doses of about 1 mg/kg of a solubleTRAIN-R are expected to be suitable starting points for optimizingtreatment dosages.

Determination of a therapeutically effective dose can also be assessedby performing in vitro experiments that measure the concentration of theblocking or activating agent. The binding assays described herein areuseful, as are other assays known in the art.

Administration of the soluble activating or blocking agents of theinvention, alone or in combination, including isolated and purifiedforms, their salts, or pharmaceutically acceptable derivative thereofmay be accomplished using any of the conventionally accepted modes ofadministration of agents which exhibit immunosuppressive activity.

EXAMPLES

Generation of Soluble Receptor Forms:

To form an receptor inhibitor for use in man, one requires the humanreceptor cDNA sequence of the extracellular domain. If the mouse form isknown, human CDNA libraries can be easily screened using the mouse cDNAsequence and such manipulations are routinely carried out in this area.With a human cDNA sequence, one can design oligonucleotide primers toPCR amplify the extracellular domain of the receptor in the absence ofthe transmembrane and intracellular domains. Typically, one includesmost of the amino acids between the last disulfide linked “TNF domain”and the transmembrane domain. One could vary the amount of “stalk”region included to optimize the potency of the resultant solublereceptor. This amplified piece would be engineered to include suitablerestriction sites to allow cloning into various C-terminal Ig fusionchimera vectors. Alternatively, one could insert a stop signal at the 3′end and make a soluble form of the receptor without resorting to the useof a Ig fusion chimera approach. The resultant vectors can be expressedin most systems used in biotechnology including yeast, insect cells,bacteria and mammalian cells and examples exist for all types ofexpression. Various human Fc domains can be attached to optimize oreliminate FcR and complement interactions as desired. Alternatively,mutated forms of these Fc domains can be used to selectively remove FcRor complement interactions or the attachment of N-linked sugars to theFc domain which has certain advantages.

Generation of Agonistic or Antagonistic Antibodies:

The above described soluble receptor forms can be used to immunize miceand to make monoclonal antibodies by conventional methods. The resultantmAbs that were identified by ELISA methods can be further screened foragonist activity either as soluble antibodies or immobilized on plasticin various in vitro cellular assays. Often the death of the HT29 cellline is a convenient system that is sensitive to signalling through manyTNF receptors. If this line does not possess the receptor of interest,that full length receptor can be stably transfected into the HT29 lineto now allow the cytotoxicity assay to work. Alternatively, such cellscan be used in the Cytosensor apparatus to assess whether activation ofthe receptor can elicit a pH change that is indicative of a signallingevent. TNF family receptors signal well in such a format and this methoddoes not require one to know the actual biological events triggered bythe receptor. The agonistic mAbs would be “humanized” for clinical use.This procedure can also be used to define antagonistic mAbs. Such mAbswould be defined by the lack of agonist activity and the ability toinhibit receptor-ligand interactions as monitored by ELISA, classicalbinding or BIAcore techniques. Lastly, the induction of chemokinesecretion by various cells in response to an agonist antibody can form ascreening assay.

Screening for Inhibitors of the Receptor-Ligand Interaction:

Using the receptor-Ig fusion protein, one can screen eithercombinatorial libraries for molecules that can bind the receptordirectly. These molecules can then be tested in an ELISA formatted assayusing the receptor-Ig fusion protein and a soluble form of the ligandfor the ability to inhibit the receptor-ligand interaction. This ELISAcan be used directly to screen various natural product libraries etc.for inhibitory compounds. The receptor can be transfected into a cellline such as the HT29 line to form a biological assay (in this casecytotoxicity) that can then form the screening assay.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the polypeptides,compositions and methods of the invention without departing from thespirit or scope of the invention. Thus, it is intended that the presentinvention cover the modifications and variations of this inventionprovided that they come within the scope of the appended claims andtheir equivalents.

Human TRAIN Receptor Identification:

Human TRAIN-R was cloned from two cDNA sequences. The first sequence(hTrainR) SEQ ID NO. 7 is a composite of two overlapping lambda gt10clones (GJ159 and GJ158) from a Clontech Humann adult lung cDNA library.The composite sequence in SEQ. ID. NO. 7 is 2185 nucleotides in lengthand encodes a 417 amino acid protein (SEQ. ID. NO.3) which has a signalsequence, a 140 amino acid extracellular domain, a transmembrane domainand a 227 amino acid intracellular domain and a stop codon. The includesanother 1200 bp. The extracellular domain of human TRAIN-R encodes threeTNF receptor like domains (it appears to be missing domain 1 whencompared to TNF-R). The sequence in SEQ. ID. NO. 3 is 19% identical tothat of low affinity nerve growth factor (LNGFR) and 24% identical toTramp/Lard4/Ws1/Dr3, both of which are members of the TNF family.

Human TRAIN-R was also cloned from a second sequence subclone of alambda gt10 cDNA (GJ156, a 790 bp subclone). The resulting sequence isshown in SEQ. ID. NO. 8. It contains intron sequence, an exon encodingthe Carboxy-terminal 30 amino acids of a secreted form of human TrainR,a stop codon and a 3′UTR. The 30 amino acids in the exon sequence were100% homologous to the murine C-term secreted form (short form of murineTrain Receptor).

Two predominant messages are observed 5 kb and 0.5 kb.

SEQ ID NO: 1   1 MALKVLPLHR TVLFAAILFL LHLACKVSCE TGDCRQQEFK DRSGNCVLCK 51 QCGPGMELSK ECGFGYGEDA QCVPCRPHRF KEDWGFQKCK PCADCALVNR 101FQRANCSHTS DAVCGDCLPG FYRKTKLVGF QDMECVPCGD PPPPYEPHCE SEQ ID NO: 2   1MALKVLPLHR TVLFAAILFL LHLACKVSCE TGDCRQQEFK DRSGNCVLCK  51 QCGPGMELSKECGFGYGEDA QCVPCRPHRF KEDWGFQKCK PCADCALVNR 101 FQRANCSHTS DAVCGDCLPGFYRKTKLVGF QDMECVPCGD PPPPYEPHCT 151 SKVNLVKISS TVSSPRDTAL AAVICSALATVLLALLILCV IYCKRQFMEK 201 KPSCKLPSLC LTVK SEQ ID NO: 3MALKVLLEQEKTFFTLLVLLGYLSCKVTCESGDCRQQEFRDRSGNCVPCNQCGPGMELSKECGFGYGEDAQCVTCRLHRFKEDWGFQKCKPCLDCAVVNRFQKANCSATSDAICGDCLPGFYRKTKLVGFQDMECVPCGDPPPPYEPHCASKVNLVKIASTASSPRDTALAAVICSALATVLLALLILCVIYCKRQFMEKKPSWSLRSQDIQYNGSELSCFDRPQLHEYAHRACCQCRRDSVQTCGPVRLLPSMCCEEACSPNPATLGCGVHSAASLQARNAGPAGEMVPTFFGSLTQSICGEFSDAWPLMQNPMGGDNISFCDSYPELTGEDIHSLNPELESSTSLDSNSSQDLVGGAVPVQSHSENFTAATDLSRYNNTLVESASTQDALTMRSQLDQ ESGAVIHPATQTSLQEA SEQID NO: 4 FYRKTKLVGFQDMECVPCGDPPPPYEPHCE* SEQ ID NO: 5   1 GGCACGAGGGCGTTTGGCGC GGAAGTGCTA CCAAGCTGCG GAAAGCGTCA  51 GTCTGGAGCA CAGCACTGGCGAGTAGCAGG AATAAACACG TTTGGTGAGA 101 GCCATGGCAC TCAAGGTCCT ACCTCTACACAGGACGGTGC TCTTCGCTGC 151 CATTCTCTTC CTACTCCACC TGGCATGTAA AGTGAGTTGCGAAACCGGAG 201 ATTGCAGGCA GCAGGAATTC AAGGATCGAT CTGGAAACTG TGTCCTCTGC251 AAACAGTGCG GACCTGGCAT GGAGTTGTCC AAGGAATGTG GCTTCGGCTA 301TGGGGAGGAT GCACAGTGTG TGCCCTGCAG GCCGCACCGG TTCAAGGAAG 351 ACTGGGGTTTCCAGAAGTGT AAGCCATGTG CGGACTGTGC GCTGGTGAAC 401 CGCTTTCAGA GGGCCAACTGCTCACACACC AGTGATGCTG TCTGCGGGGA 451 CTGCCTGCCA GGATTTTACC GGAAGACCAAACTGGTTGGT TTTCAAGACA 501 TGGAGTGTGT GCCCTGCGGA GACCCACCTC CTCCCTACGAACCACACTGT 551 GAGTGATGTG CCAAGTGGCA GCAGACCTTT AAAAAAAAAA GAAAAAAAA SEQID NO: 6   1 CGGCACGAGG GCCGGCACCC CGCGCCACCC CAGCCTCAAA CTGCAGTCCG  51GCGCCGCGGG GCAGGACAAG GGGAAGGAAT AAACACGTTT GGTGAGAGCC 101 ATGGCACTCAAGGTCCTACC TCTACACAGG ACGGTGCTCT TCGCTGCCAT 151 TCTCTTCCTA CTCCACCTGGCATGTAAAGT GAGTTGCGAA ACCGGAGATT 201 GCAGGCAGCA GGAATTCAAG GATCGATCTGGAAACTGTGT CCTCTGCAAA 251 CAGTGCGGAC CTGGCATGGA GTTGTCCAAG GAATGTGGCTTCGGCTATGG 301 GGAGGATGCA CAGTGTGTGC CCTGCAGGCC GCACCGGTTC AAGGAAGACT351 GGGGTTTCCA GAAGTGTAAG CCATGTGCGG ACTGTGCGCT GGTGAACCGC 401TTTCAGAGGG CCAACTGCTC ACACACCAGT GATGCTGTCT GCGGGGACTG 451 CCTGCCAGGATTTTACCGGA AGACCAAACT GGTTGGTTTT CAAGACATGG 501 AGTGTGTGCC CTGCGGAGACCCACCTCCTC CCTACGAACC ACACTGTACC 551 AGCAAGGTGA ACCTTGTGAA GATCTCCTCCACCGTCTCCA GCCCTCGGGA 601 CACGGCGCTG GCTGCCGTCA TCTGCAGTGC TCTGGCCACGGTGCTGCTCG 651 CCCTGCTCAT CCTGTGTGTC ATCTACTGCA AGAGGCAGTT CATGGAGAAG701 AAACCCAGCT GTAAGCTCCC ATCCCTCTGT CTCACTGTGA AGTGAGCTTG 751TTAGCATTGT CACCCAAGAG TTCTCAAGAC ACCTGGCTGA GACCTAAGAC 801 CTTTAGAGCATCAACAGCTA CTTAGAATAC AAGATGCAGG AAAACGAGCC 851 TCTTCAGGAA TCTCAGGGCCTCCTAGGGAT GCTGGCAAGG CTGTGATGTC 901 TCAAGCTACC AGGAAAAATT TAAAGTTGTTTWTCCCCTAA AA SEQ ID NO: 7GAATTCCGGGGGAGGTGCACGGTGTGCACGCTGGACTGGACCCCCCATGCAACCCCGCGCCCTGCGCCTTAACCAGGACTGCTCCGCGCGCCCCTGAGCCTCGGGCTCCGGCCCGGACCTGCAGCCTCCCAGGTGCCTGGGAAGAACTCTCCAACAATAAATACATTTGATAAGAAAGATGGCTTTAAAAGTGCTACTAGAACAAGAGAAAACGTTTTTCACTCTTTTAGTATTACTAGGCTATTTGTCATGTAAAGTGACTTGTGAATCACGAGACTGTAGACAGCAAGAATTCAGGGATCGGTCTGGAAACTGTGTTCCCTGCAACCAGTGTGGGCCAGGCATGGAGTTGTCTAAGGAATGTGGCTTCGGCTATGGGGAGGATGCACAGTGTGTGACGTGCCGGCTGCACAGGTTCAAGGAGGACTGGGGCTTCCAGAAATGCAAGCCCTGTCTGGACTGCGCAGTGGTGAACCGCTTTCAGAAGGCAAATTGTTCAGCCACCAGTGATGCCATCTGCGGGGACTGCTTGCCAGGATTTTATAGGAAGACGAAACTTGTCGGCTTTCAAGACATGGAGTGTGTGCCTTGTGGAGACCCTCCTCCTCCTTACGAACCGCACTGTGCCAGCAAGGTCAACCTCGTGAAGATCGCGTCCACGGCCTCCAGCCCACGGGACACGGCGCTGGCTGCCGTTATCTGCAGCGCTCTGGCCACCGTCCTGCTGGCCCTGCTCATCCTCTGTGTCATCTATTGTAAGAGACAGTTTATGGAGAAGAAACCCAGCTGGTCTCTGCGGTCGCAGGACATTCAGTACAACGGCTCTGAGCTGTCGTGTTTTGACAGACCTCAGCTCCACGAATATGCCCACAGAGCCTGCTGCCAGTGCCGCCGTGACTCAGTCCAGACCTGCGGGCCGGTGCGCTTGCTCCCATCCATGTGCTGTGAGGAGGCCTGCAGCCCCAACCCGGCGACTCTTGGTTGTGGGGTGCATTCTGCAGCCAGTCTTCAGGCAAGAAACGCAGGCCCAGCCGGGGAGATGGTGCCGACTTTCTTCGGATCCCTCACGCAGTCCATCTGTGGCGAGTTTTCAGATGCCTGGCCTCTGATGCAGAATCCCATGGGTGGTGACAACATCTCTTTTTGTGACTCTTATCCTGAACTCACTGGAGAAGACATTCATTCTCTCAATCCAGAACTTGAAAGCTCAACGTCTTTGGATTCAAATACCAGTCAAGATTTGGTTGGTGGGGCTGTTCCAGTCCAGTCTCATTCTGAAAACTTTACAGCAGCTACTGATTTATCTAGATATAACAACACACTOGTAGAATCAGCATCAACTCAGGATGCACTAACTATGAGAAGCCAGCTAGATCAGGAGAGTGGCGCTGTCATCCACCCAGCCACTCAGACGTCCCTCCAGGAAGCTTAAAGAACCTGCTTCTTTCTGCAGTAGAAGCGTGTGCTGGAACCCAAAGAGTACTCCTTTGTTAGGCTTATGGACTGAGCAGTCTGGACCTTGCATGGCTTCTGGGGCAAAAATAAATCTGAACGAAACTGACGGCATTTGAAGCCTTTCAGCCAGTTGCTTCTGAGCCAGACCAGCTGTAAGCTGAAACCTCAATGAATAACAAGAAAAGACTCCAGGCCGACTCATGATACTCTGCATCTTTCCTACATGAGAAGCTTCTCTGCCACAAAAGTGACTTCAAAGACGGATGGGTTGAGCTGGCAGCCTATGAGATTGTGGACATATAACAAGAAACAGAAATGCCCTCATGCTTATTTTCATGGTGATTGTGGTTTTACAAGACTGAAGACCCAGAGTATACTTTTTCTTTCCAGAAATAATTTCATACCGCCTATGAAATATCAGATAAATTACCTTAGCTTTTATGTAGAATGGGTTCAAAAGTGAGTGTTTCTATTTGAGAAGGACACTTTTTCATCATCTAAACTGATTCGCATAGGTGGTTAGAATGGCCCTCATATTGCCTGCCTAAATCTTGGGTTTATTAGATGAAGTTTACTGAATCAGAGGAATCAGACAGAGGAGGATAGCTCTTTCCAGAATCCACACTTCTGACCTCAGCCTCGGTCTCATGAACACCCGCTGATCTCAGGAGAACACCTGGGCTAGGGAATGTGGTCGAGAAAGGGCAGCCCATTGCCCAGAATTAACACA SEQ ID NO: 8GAATTCCAAATGCTAAAACCTAGTTCTTTATTCATCTATAAGGTATTTTGTCGTTTAAGTTTCAATAAAAATGCCGAAGACCACTGACTTTATATTCCCCCACCTGCACCCCCACCCCAATATAGAAGAAGTGCACTGAGAAGCATCTGCAAAGTTAGCTTTAGGGGAATTGATATTTCTTAAGTGTCCACTGCTTCCTCTTCAAAAATGTGTCTACCTAAGATACTATTATTTAAGCCTCTGTGTACTTTTAACCGTAGAACTGGTAATGGAGACTGCTGGTAATTTATGACCACAACTGTAAGCTTAGATGAAAGAGTTAACAAGGAGTATTTTCCTTTCTCTTCTAGATTTTATAGGAAGACGAAACTTGTCGGCTTTCAAGACATGGAGTGTGTGCCTTGTGGAGACCCTCCTCCTCCTTACGAACGGCACTGTGAGTGAACGCAACACAGGCAGAGCCAAGGGGACGCCTGGCCTTTTGAAAAAGTTTAAATTTGTAAACGTTTCTTCTCTGGCAGATGGACCCAAATCTGTCTCTCCTGTGGGGTGTACAGTGTGTCCTCTTTAATCAGGCTTCTGGCAGGACAGAAAGTCCCTTTGTTCTGTGCCTCAGTCAGCAAACCGGTCCCAGGGATTTGAATCTCAGAGTGGAGTGCAGACATTTTGCCACTGCTCAGCTCCTTCTGAAGCCTTCCCTGGCACCCTGGGTCTGTAATTCAGGCCACTTTGAATAACCAGGCGGCTCACATCCTCACTCTTAGGTCTTCGTGCCCTGGCCCCATGAATTC SEQ ID. NO. 9MALKVLLEQEKTFFTLLVLLGYLSCKVTCESGDCRQQEFRDRSGNCVPCNQCGPGMELSKECGFGYGEDAQCVTCRLHRFKEDWGFQKCKPCLDCAVVNRFQKANCSATSDAICGDCLPGFYRKTKLVGFQDMECVPCGDPPPPYEPHCE

1. An isolated antibody that specifically binds to a human TNF Receptorin the BRAIN (TRAIN) polypeptide comprising an amino acid sequenceselected from the group consisting of: (a) SEQ ID NO: 3; (b) SEQ ID NO:9; (c) amino acid residues 26 to 417 of SEQ ID NO: 3; (d) amino acidresidues 1 to 173 of SEQ ID NO: 3; (e) amino acid residues 26 to 173 ofSEQ ID NO: 3; (f) amino acid residues 174 to 190 of SEQ ID NO: 3; (g)amino acid residues 191 to 417 of SEQ ID NO:
 3. 2. The antibody of claim1, wherein the antibody is a monoclonal antibody.
 3. The monoclonalantibody of claim 2, wherein the antibody is a chimeric or humanizedantibody comprising human constant regions.
 4. The antibody of claim 1,wherein the antibody inhibits TRAIN receptor signaling.
 5. The antibodyof claim 1, wherein the antibody activates TRAIN receptor signaling. 6.A pharmaceutical composition comprising the antibody of claim 1 and apharmaceutically acceptable carrier.
 7. A pharmaceutical compositioncomprising the antibody of claim 2 and a pharmaceutically acceptablecarrier.
 8. A pharmaceutical composition comprising the antibody ofclaim 3 and a pharmaceutically acceptable carrier.
 9. A pharmaceuticalcomposition comprising the antibody of claim 4 and a pharmaceuticallyacceptable carrier.
 10. A pharmaceutical composition comprising theantibody of claim 5 and a pharmaceutically acceptable carrier.
 11. Amethod of treating a tumor in a patient wherein the tumor selectivelyexpresses human TRAIN receptor, comprising the step of administering tothe patient an amount of the antibody of claim 1 effective to treat thetumor.